Chloride channels have important roles in normal human physiology, and their dysfunction is involved in a number of diseases. An important example of a mis-functioning chloride channel is mutation of the cystic fibrosis transmembrane conductance regulator (CFTR), which causes cystic fibrosis (CF), one of the most common life-limiting genetic diseases. Another class of chloride channels are the calcium-activated chloride channels (CaCCs), such as TMEM16A, which are postulated to be drug targets for human diseases. Inhibition of CaCCs has been suggested as a method to treat hypertension, pain, diarrhea, and excess mucus production. Since our first submission of this grant, TMEM16a inhibitors have been experimentally shown to be promising candidates for the treatment of hypertension and asthma. Furthermore, recent evidence suggests a link between TMEM16a expression and the proliferation of prostate and pancreatic cancer. In this proposal, we will systematically explore the SAR of the modular 2-amino-4-arylthiazole scaffold for its efficacy to inhibit the TMEM16A, the first characterized CaCC. The central hypotheses of this proposal are: (a) the molecular structure of the 2-amino-4-arylthiazoles can be modified at several positions to alter the interactions with TMEM16A, in turn improving inhibitory potency; (b) given libraries of TMEM16a modulators, computational pharmacophore models for inhibitors can be created which will facilitate further optimization and can also serve as a template for virtual screening; (c) the binding mode is specific and can be predicted through homology modeling and confirmed with photoaffinity labeling. Successful completion of this project will fill a gap in the knowledge of CaCC-TMEM16A, namely the successful generation of novel inhibitors based on a very promising, and relatively potent HTS lead scaffold. Such inhibitors, which we anticipate can be optimized into the nanomolar range, could serve in future drug development studies, and could also contribute to the basic scientific understanding of TMEM16A as a drug target in specific, and CaCCs in general. Herein, we are pursuing three specific aims, having to do with the systematic optimization of the 2-amino-4- arylthiazole scaffold: (1) explore the effect of the 2-pyrimidine heterocycle on the potency of TMEM16A inhibitors; (2) explore the effect of the thiazole-conjugated aryl (or heteroaryl) ring systems on the potency of TMEM16A inhibitors; generation of a library of synergistic inhibitors; (3) generation of pharmacophore models for inhibitors of TMEM16a; prediction of the mode of binding using homology modeling; and confirmation of the binding mode by photoaffinity labeling.